Breast cancer-related death results from complications due to metastatic growth. Therapies for metastatic patients focus on extending survival and controlling symptoms, and the choice of therapies is frequently based on particular target-proteins found in the primary breast tumor, which is usually excised years before development of metastatic disease. Therapies for these target-proteins are much more effective in treating metastatic patients than standard chemotherapy. Because cancer changes over the time, the target-proteins in some metastatic tumors could well have changed from levels on the primary tumor. Biopsies that could test target proteins levels in metastatic tissue pose a problem because they are invasive, pose risk to the patient and are infeasible for patients with multiple different tumor sites. This proposal addresses locating tumor cells circulating in the blood and testing them for the target-proteins that help selection of a therapy. Because, circulating tumor cells (CTCs) represent the current metastatic disease, they arguably better represent metastatic disease than the primary tumor. Circulating tumor cells are rare and difficult to find. Current technologies used to find CTCs have problems. Some miss certain types of tumor cells due to low levels of expression of the enrichment target and provide poor images of the cells making it hard to be sure that they are CTCs. Others rely on size to find CTCs, but size varies widely, and such technologies miss small CTCs. In this work a research instrument, the FAST cytometer, uses an enrichment target that is known to be expressed at high levels in solid tumor cells. The assay does not degrade the cell morphology, and imaging is done on a planar surface for high fidelity. This proposal addresses the work needed to transform the research instrument into an instrument approved for use in a clinical trial. Methods will be developed to monitor the accuracy of the instrument and the multiplex assay. Procedures will be established to enable the CLIA certification needed for the clinical trial. The clinical trial will focus on metastatic breast cancer patients who have a triple negative primary tumor. Patients with this tumor type have no target-proteins and are ineligible to receive the targeted therapies. Consequently, they face a much poorer outcome. Preliminary data with a multiplex assay for 3 target-proteins has shown that target-proteins on CTCs are present in a significant number of patients. The clinical trial in Phase II will determine if these patients survive longer when the relevant target-therapies are administered. Clinical validation of the use of target protein levels on CTCs should enable more effective and personalized targeted therapy and prevention of acquired drug resistance, resulting in better response, improved progression-free and better overall survival. PUBLIC HEALTH RELEVANCE: Metastatic breast cancer can be optimally treated with special therapies that are selected by the presence of related proteins in the primary breast tumor, which is usually excised years before development of metastatic disease. As cancer is known to change over the time, the proteins in metastatic tumors could well be different. This proposal seeks to locate tumor cells circulating in the blood and determine in a clinical trial whether their proteins can better guide the selection of a therapy. The trial will focus on patients who have no special proteins on their primary tumors and hence face a worse outcome.